Plasmid encoding IAA and a method thereof

ABSTRACT

The present invention relates to a plasmid pUPI126 encoding indole-3 acetic acid (IAA) production; it also relates to a  Acinetobacter  strains having plasmid pUPI126; a bioinoculum for promoting growth of wheat plant, and lastly, it relates to a method of promoting wheat plant growth, said method comprising treating wheat seeds with the bioinoculum.

FIELD OF THE INVENTION

The present invention relates to a plasmid pUPI126, encoding indole-3 acetic acid (IAA) production; it also relates to an Acinetobacter strains having plasmid pUPI126; a bioinoculum for promoting growth of wheat plant, and lastly, it relates to a method of promoting wheat plant growth, said method comprising treating wheat seeds with the bioinoculum.

BACKGROUND OF THE INVENTION

Acinetobacter species are ubiquitous in nature (Baumann, 1968; Juni, 1972,). Acinetobacter is commonly found in soil, water, food and also present on healthy human skin.(Saha & Chopade, 2001; Patil & Chopade 2001; Dhakephalkar et al, 1994a). Acinetobacter is one of the known opportunistic human pathogens (Dhakephalkar & Chopade 1994a; Chopade et al, 1994a & 1994b). It also possesses number of naturally occurring plasmids exhibiting resistance to antibiotics and heavy metals (Deshpande et al 1994; Shakibaie et al, 1999). There are few reports on presence of Acinetobacter in soil, however, detail studies regarding its occurrence, distribution, growth pattern, physiology and its interactions with other soil microorganisms are not known. There is only one statement on presence of Acinetobacter in wheat rhizosphere (Kleeberger et al, 1983). However, detail information about role of Acinetobacter in rhizosphere is not known.

Soil is a rich environment for growth of microorganisms and specifically rhizosphere is highly specialized environment in soil for growth of microorganisms. Since rhizosphere contains large number of microorganisms one would expect plasmid transfer and dynamics of plasmid transfer from Acinetobacter to other microorganisms and vice versa in the rhizosphere environment. The rhizosphere of each and every plant is very specific with respect to the root exudates as it is the main source of nutrients for rhizosphere microorganisms (Subba Rao, 1986).

Until now there is no report on involvement of plasmid/s in the production of IAA from genus Acinetobacter. IAA is one of the major plant growth promoting hormones produced by plants as well as some bacteria and fungi. (Arshad & Frankenberger,1991). Many species of bacteria produce IAA, especially when growth media are supplemented with tryptophan, a precursor of IAA. Number of microorganisms like Agrobacterium tumefaciens, Agrobacterium rhizogenes, Pseudomonas savastanoi, Pseudomonas spp.,(Leinhos & Vocek, 1984), Rhizobium spp, (Baldi et al, 1991), Bradyrhizobium spp. and Azospirillum spp (Bashan et al, 1989) present in the rhizosphere of plants are known to produce IAA (Costacurta & Vanderleyden,1995). To some extent the biosynthesis of plant growth promoting substances like auxin/s from phosphate solubilizing rhizobacteria from rhizosphere of wheat and rye is reported (Leinhos & Vocek, 1984). Aim of this work was to isolate and characterize Acinetobacter from rhizosphere of wheat and to find out the role of Acinetobacter in plant growth promotion in general and involvement of plasmid/s in production of plant growth promoting substance like indole acetic acid (IAA). Acinetobacters were isolated from rhizosphere of wheat. The variety of wheat plant was HD 2189 ICAR, New Delhi, India. The rhizosphere soil was collected throughout December to March 1998, growing season, at the different stages of life cycle of wheat plant: control soil (0 d), elongation (30 d) flowering stage (45 d), fruiting stage (60 d) and ripened fruiting stage (75 d) from three areas—rhizosphere soil (RS), rhizoplane (RP), and non rhizosphere soil (NRS). The samples were collected from an agricultural field of Mahatma Phule Agriculture College, Shivajinagar, Pune, Maharashtra, India. Samples were brought to laboratory and processed immediately within half an hour.

OBJECTS OF THE PRESENT INVENTION

The main object of the present invention is to develop a plasmid encoding indole-3 acetic acid (IAA) production.

Another main object of the present invention relates to Acinetobacter strains encoding indole-3 acetic acid (IAA) production.

Yet another object of the present invention relates to developing a bioinoculum for promoting growth of wheat plant.

Still another object of the present invention relates to a method of promoting wheat plant growth.

SUMMARY OF THE PRESENT INVENTION

The present invention relates to a plasmid pUPI126 of encoding indole-3 acetic acid (IAA) production; it also relates to a Acinetobacter strains having plasmid pUPI126; a bioinoculum for promoting growth of wheat plant, and lastly, it relates to a method of promoting wheat plant growth, said method comprising treating wheat seeds with the bioinoculum.

DESCRIPTION OF THE INVENTION

Accordingly, the present invention relates to a plasmid pUPI126 of encoding indole-3 acetic acid (IAA) production; it also relates to a Acinetobacter strains having plasmid pUPI126; a bioinoculum for promoting growth of wheat plant, and lastly, it relates to a method of promoting wheat plant growth, said method comprising treating wheat seeds with the bioinoculum.

In yet another embodiment of the present invention, wherein a plasmid pUPI126 encoding indole-3 acetic acid (IAA) production.

In still another embodiment of the present invention, wherein the plasmid is of 40 Kb.

In yet another embodiment of the present invention, wherein the plasmid encodes resistance to selenium, tellurium, and lead.

One more embodiment of the present invention, Acinetobacter strains having plasmid pUPI126 encoding indole-3 acetic acid (IAA) production.

In still another embodiment of the present invention, wherein, the strains as claimed in claim 3, wherein the strains are selected from a group comprising Acinetobacter haemolyticus A19, Acinetobacter genospecies A28, Acinetobacter genospecies A15, Acinetobacter baumannii A13, Acinetobacter baumannii A16, Acinetobacter baumannii A18, Acinetobacter baumannii A30, and Acinetobacter junii A6.

In yet another embodiment of the present invention, wherein the plasmid encodes resistance to selenium, tellurium, and lead.

In still another embodiment of the present invention, wherein the strain Acinetobacter haemolyticus A19 is characterized as: Characteristics A. haemolytivusA19 Growth at: 44⁰ C. ++ 41⁰ C. ++ 37⁰ C. ++ 28⁰ C. ++ Pigment production Yellow Acid from Glucose ++ Gelatin Hydrolysis ++ Haemolysis ++ Citrate (Simmons) ++ Utilization of: DL - Lactate ++ L - Phenylalanine ++ Phenyl acetate ++ Malonate ++ L - Histidine ++ D - Malate ++ L - Aspartate −− L - Leucine −− L - Tyrosine ++ β- Alanine ++ L - Glycine ++ Trans-Aconitate −− D- Glucose ++ L - Tryptophane ++ Na - Acetate ++ Oxalate ++ Ethanol ++ L - Arginine ++ L - Ornithine ++ DL 4-Aminobutyrate ++ Tween 20 ++ Tween 80 ++ Other tests: Indole −− MR −− VP −− Triple sugar iron test ++

In yet another embodiment of the present invention, wherein the strain Acinetobacter genospecies A28 is characterized as: Characteristics A. genospecies 3 A28 Growth at: 44⁰ C. −− 41⁰ C. ++ 37⁰ C. ++ 28⁰ C. ++ Pigment production Pink Acid from Glucose ++ Gelatin Hydrolysis −− Haemolysis −− Citrate (Simmons) ++ Utilization of: DL - Lactate ++ L - Phenylalanine ++ Phenyl acetate −− Malonate ++ L - Histidine ++ D - Malate ++ L - Aspartate ++ L - Leucine ++ L - Tyrosine ++ β- Alanine ++ L - Glycine ++ Trans-Aconitate ++ D- Glucose ++ L - Tryptophane ++ Na - Acetate ++ Ethanol ++ L - Arginine ++ L - Ornithine ++ DL 4-Aminobutyrate ++ Other tests: MR ++ VP −− Triple sugar ion test ++

In still another embodiment of the present invention, the strain Acinetobacter genospecies a15 is characterized as: Characteristics A. genospecies A15 Growth at: 44⁰ C. −− 41⁰ C. ++ 37⁰ C. ++ 28⁰ C. ++ Pigment production Pink Acid from Glucose ++ Gelatin Hydrolysis −− Haemolysis −− Citrate (Simmons) ++ Utilization of: DL - Lactate ++ L - Phenylalanine ++ Phenyl acetate ++ Malonate ++ L - Histidine ++ D - Malate ++ L - Aspartate ++ L - Leucine ++ L - Tyrosine ++ β- Alanine ++ L - Glycine ++ Trans-Aconitate ++ D- Glucose ++ L - Tryptophane ++ Na - Acetate ++ Ethanol ++ L - Arginine ++ L - Ornithine ++ DL 4-Aminobutyrate ++ Other tests: MR ++ VP −− Triple sugar ion test ++

In yet another embodiment of the present invention, wherein the strain Acinetobacter baumannii A13 is characterized as: Character A. baumannii A13 Growth at: 44⁰ C. ++ 41⁰ C. ++ 37⁰ C. ++ 28⁰ C. ++ Pigment production NP Acid from Glucose ++ Gelatin Hydrolysis −− Haemolysis ++ Citrate (Simmons) ++ Utilization of: DL - Lactate ++ L - Phenylalanine ++ Phenyl acetate ++ Malonate ++ L - Histidine ++ D - Malate ++ L - Aspartate ++ L - Leucine ++ L - Tyrosine ++ β- Alanine ++ L - Glycine ++ Trans-Aconitate −− D- Glucose ++ L - Tryptophane ++ Na - Acetate ++ Ethanol ++ L - Arginine ++ L - Ornithine ++ DL 4-aminobutyrate ++ Other tests: Indole −− MR −− Triple sugar iron test ++

In still another embodiment of the present invention, wherein the strain Acinetobacter baumannii A16 is characterized as: Character A. baumannii A16 Growth at: 44⁰ C. ++ 41⁰ C. ++ 37⁰ C. ++ 28⁰ C. ++ Pigment production Brown Acid from Glucose NP Gelatin Hydrolysis −− Haemolysis −− Citrate (Simmons) ++ Utilization of: DL - Lactate ++ L - Phenylalanine ++ Phenyl acetate −− Malonate ++ L - Histidine ++ D - Malate ++ L - Aspartate ++ L - Leucine ++ L - Tyrosine ++ β- Alanine ++ L - Glycine ++ Trans-Aconitate ++ D- Glucose ++ L - Tryptophane ++ Na - Acetate ++ Ethanol ++ L - Arginine ++ L - Ornithine ++ DL 4-aminobutyrate ++ Other tests: Indole −− MR −− Triple sugar iron test ++

In yet another embodiment of the present invention, wherein the strain Acinetobacter baumannii A18 is characterized as: Character A. baumannii A18 Growth at: 44⁰ C. ++ 41⁰ C. ++ 37⁰ C. ++ 28⁰ C. ++ Pigment production Yellow Acid from Glucose ++ Gelatin Hydrolysis −− Haemolysis −− Citrate (Simmons) ++ Utilization of: DL - Lactate ++ L - Phenylalanine ++ Phenyl acetate ++ Malonate ++ L - Histidine ++ D - Malate −− L - Aspartate ++ L - Leucine ++ L - Tyrosine ++ β- Alanine ++ L - Glycine ++ Trans-Aconitate ++ D- Glucose ++ L - Tryptophane ++ Na - Acetate ++ Ethanol ++ L - Arginine ++ L - Ornithine −− DL 4-aminobutyrate ++ Other tests: Indole −− MR −− Triple sugar iron test ++

In still another embodiment of the present invention, wherein the strain Acinetobacter baumannii A30 is characterized as: Character A. baumannii A30 Growth at: 44⁰ C. ++ 41⁰ C. ++ 37⁰ C. ++ 28⁰ C. ++ Pigment production NF Acid from Glucose NP Gelatin Hydrolysis ++ Haemolysis ++ Citrate (Simmons) ++ Utilization of: DL - Lactate ++ L - Phenylalanine −− Phenyl acetate −− Malonate ++ L - Histidine ++ D - Malate ++ L - Aspartate −− L - Leucine −− L - Tyrosine −− β- Alanine ++ L - Glycine ++ Trans-Aconitate −− D- Glucose ++ L - Tryptophane ++ Na - Acetate ++ Ethanol ++ L - Arginine ++ L - Ornithine ++ DL 4-aminobutyrate ++ Other tests: Indole −− MR ++ Triple sugar iron test ++

In yet another embodiment of the present invention, wherein the strain Acinetobacter junii A6 is characterized as: Characteristics A. junii A6 Growth at: 44⁰ C. ++ 41⁰ C. ++ 37⁰ C. ++ 28⁰ C. ++ Pigment production −− Acid from Glucose −− Gelatin Hydrolysis ++ Haemolysis −− Citrate (Simmons) ++ Utilization of: DL - Lactate ++ L - Phenylalanine ++ Phenyl acetate ++ Malonate ++ L - Histidine ++ D - Malate −− L - Aspartate ++ L - Leucine ++ L - Tyrosine ++ β- Alanine ++ L - Glycine ++ Trans-Aconitate ++ D- Glucose ++ L - Tryptophane ++ Na - Acetate ++ Oxalate −− Ethanol ++ L - Arginine ++ L - Ornithine −− DL 4-Aminobutyrate ++ Tween 20 ++ Tween 80 ++ Other tests: Indole −− MR −− VP −− Triple sugar iron test ++

Another embodiment of the present invention, wherein a bioinoculum for wheat plant, said bioinoculum comprising one or more strain selected from a group consisting of Acinetobacter haemolyticus A19, Acinetobacter genospecies A28, Acinetobacter genospecies A15, Acinetobacter baumannii A13, Acinetobacter baumannii A16, Acinetobacter baumannii A18, Acinetobacter baumannii A30, and Acinetobacter junii A6; and a carrier.

In still another embodiment of the present invention, wherein the carrier is lignite. One more embodiment of the present invention, wherein the said method comprising treating wheat seeds with the bioinoculum.

In yet another embodiment of the present invention, wherein the bioinoculum comprising one or more strain selected from a group consisting of Acinetobacter haemolyticus A19, Acinetobacter genospecies A28, Acinetobacter genospecies A15, Acinetobacter baumannii A13, Acinetobacter baumannii A16, Acinetobacter baumannii A18, Acinetobacter baumannii A30, and Acinetobacter junii A6; and a carrier.

In still another embodiment of the present invention, wherein the carrier is lignite. In yet another embodiment of the present invention, wherein the Acinetobacter colonizes during the treatment of the seed.

In still another embodiment of the present invention, wherein the color of the leaf becomes darker green.

In yet another embodiment of the present invention, wherein the inflorescence and fruiting stages of the plant is reached in time duration lessened by 10 days.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1.A. IAA production by four Acinetobacter genospecies—A. baumannii A13, A. genospecies 3 A15 & A28, A. junii A6 isolated from rhizosphere of wheat.

FIG. 1.B. IAA production by four Acinetobacter genospecies A. haemolyticus A19 and A. baumannii A18, A16 and A30 isolated from rhizosphere of wheat

FIG. 2. TLC of purified IAA produced from Acinetobacter genospecies S. purified IAA; 30, A. baumannii (A30); 28, A. genospecies 3 (A28); 19, A. haemolyticus (A19); 18, A. baumannii (A18); 16, A. baumannii (A16); 15, Acinetobacter genospecies 3 (A15); 13, A. baumannii (A13); 6, A. junii (A6); I, Standard IAA.

FIGS. 3(A) and (B). (A) IR spectrum of purified IAA Acinetobacter genospecies. (B) IR spectrum of standard IAA from Sigma % T, percentage of transmission; cm⁻¹, wavelength in centimeters.

FIG. 4 ¹H-NMR analysis of purified IAA from Acinetobacter genospecies.

FIG. 5 Effect of IAA produced by Acinetobacter genospecies on growth of wheat plant (A) Root and shoot length of 21-d wheat plant; (B) shoot width of 60-d wheat plant (C) leaf width of 60-d wheat plant; (D) fruiting size andnnumber of grains of 75-d wheat plant.

FIG. 6 Plasmid pUPI126 in IAA-producing Acinetobacter genospecies. Lane 1, A. hemolyticus (A19); Lane 2, A. baumannii (A13); Lane 3, A. baumannii (A16); lane 4, Acinetobacter genospecies 3 (A15); lane 5, A. baumannii (A18); lane 6, A. A. junii (A6); lane 7 A. genospecies 3 (A28).

The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.

EXAMPLES

Culture Media:

Five different culture media were used for isolation of Acinetobacter and other gram negative bacteria from wheat rhizosphere. The media used were violet red bile agar (VRBA) (Kleeberger et al, (1983), and Cystine lactose electrolyte deficient medium (CLED, HiMedia laboratories Limited, Mumbai, India), for all gram negative bacteria, Acinetobacter minimal medium (AMM) (Juni, 1972), and Holton's selective medium (Holton, 1983), for growth of Acinetobacter and standard plate count agar (SPCA), used for estimation of total count of culturable bacteria present in all three samples (RP, RS, and NRS).

Isolation of Acinetobacter from Rhizosphere:

Dilution plate method (Subba Rao, 1986) was used for isolation of Acinetobacter and all other gram negative bacteria from wheat rhizosphere and rhizoplane. All types of colonies were isolated from four selective media and total bacterial counts were taken from SPCA. Colonies from VRBA, AMM, CLED and Holton's media were characterized by their morphological features and tentatively identified up to genus level by gram character & morphology, motility, oxidase test, catalase test and capsule staining.

Chromosomal DNA Transformation Assay:

Chromosomal DNA transformation assay was carried out using naturally competent auxotrophic mutant A. calcoaceticus BD413 trpE27. Transformation was done by modified method of Juni (In brief: Temperature used for lysis was 65° C. for 90 min.), using crude DNA (Juni,1972), as well as purified DNA (Chen & Kuo, 1993) isolated from Acinetobacter. The growth of transformants of Acinetobacter on Acinetobacter Minimal Medium (AMM) without tryptophan was considered as a positive result of DNA transformation.

Identification of Acinetobacter Strains to Species Level:

Acinetobacter strains were classified to species level by Bouvet and Grimont classification system (1986 & 1987). API 20NE was also employed for biotyping of Acinetobacter strains isolated from wheat rhizosphere (Towner & Chopade, 1987).

Detection of IAA Production in Acinetobacter spp:

IAA production was detected by two methods as described below:

Nitrocellulose Paper Assay:

All 37 Acinetobacter strains were tested by nitrocellulose paper assay (Bric et al 1991) for the production of IAA. 37 Acinetobacter strains were spot inoculated on LB medium supplemented with 5 mM tryptophan (LBT). The spot inoculated agar surface was overlaid with a nitrocellulose membrane filter and incubated at 28° C. for 48 h. The membrane filter was aseptically removed from the plate, after 48 h and transferred to Whatman filter paper No. 2 and Salkowaski reagent 500 μl (2% of 0.5M FeCl₃ in 35% perchloric acid or FeCl₃ 2.025 g in 300 ml of conc. H₂SO₄ and 500 ml of distilled water) was added on the nitrocellulose paper and kept for 1-2 min. at room temperature. IAA production was indicated by red ring around the colony.

Salkowaski Method:

In this method, (Gordons & Weber, 1951), 37 Acinetobacter spp were grown at 28° C. in the LB broth supplemented with 1 mg/ml of tryptophan. After 48 h of incubation cells were harvested by centrifugation at 10000 rpm for 15 min. at room temperature and 1 ml of sample (supernatant) and 4 ml of Salkowaski reagent (Gordons & Weber, 1951) were mixed and allowed to react in dark at room temperature for 30 min. 1 ml of uninoculated LBT and 4 ml of Salkowaski reagent was treated as blank. Optical density (O.D) was checked at 540 nm. Red color formation was considered as positive evidence for IAA production. (Bric et al, 1991).

Time Course of IAA Producing Acinetobacter spp.:

IAA production by Acinetobacter strains at different growth phases was also studied Acinetobacter strains were inoculated in LBT medium, incubated at 28° C. at 120 rpm and production of IAA was checked after every 2 h up to 108 h by Salkowaski method.

Extraction and Purification of IAA by Preparative Thin Layer Chromatography (TLC):

IAA produced by Acinetobacter genospecies was purified by method described by Koga et al (1991). In brief: all strains were grown in LBT medium till it shows maximum IAA production. 150 ml culture broth was centrifuged at 8000 rpm (Remi, RMI2C, India) for 20 min at room temperature. pH of the supernatant was adjusted to 7 (neutral extract) and it was extracted with(1:1 volume) ethyl acetate. The aqueous phase was carefully separated and pH was adjusted to 2.8 with HCl (acid extract). This acid extract was again extracted with (1:1 volume) ethyl acetate. Organic phases from both extractions were mixed together and evaporated on rota-evaporator at 60° C. (Buchi, Switzerland) to get powdered IAA. At each phase of extraction, Salkowaski test was done for organic as well as aqueous phase. The preparative TLC was run for the extracted samples with the standard indole 3 acetic acid (Sigma,USA) using methanol:chloroform (10:90) as a solvent system. The TLC was carried out on polygram G/UV 254 precoated aluminium sheet of 20×20 and 60 mm (Merck, Germany). The TLC spots were observed under UV (245 nm) and compared with standard IAA sample (Sigma, USA). IAA spots were scratched with a fine spatula and sample with silica gel was collected in a clean glass bottle. The sample was dissolved in 2 ml of chloroform or ethyl acetate and filtered through cotton to remove silica. TLC of the sample was done to check purity of the sample.

Identification of IAA by Infra Red (IR) Spectrum and Melting Point:

IR spectrum of extracted IAA sample was taken using Perkin Elmer 1600 FTIR Spectrophotometer and the spectra were recorded in nujol mull or in KBr pellets and expressed in wave number (cm⁻¹). The melting point of extracted IAA was tested on Thomos Hoover melting point apparatus in the Degree Celsius.

Analysis of IAA by ¹H-NMR:

The purified sample of IAA was analyzed by Mercury ¹H-NMR (300 MHZ, Vavion, USA) and the peaks were identified for IAA. The ¹H-NMR of standard IAA was also checked. The two ¹H-NMR were compared. The purified IAA was dissolved in 25 μl of DMSO.

Effect of pH on IAA Production:

To study effect of pH on the production of IAA buffered LBT broth was prepared in the standard buffers such as acetate, phosphate and Tris-Hcl (Gerhardt et al, 1994). pH in the range of 4 to 9 was checked. The maximum IAA production phase at different pH was checked by Salkowaski test.

Effect of IAA Production by Acinetobacter spp. on Growth of Wheat Plant:

The effect of IAA production on growth of wheat plant was tested by pot experiments. All eight Acinetobacter strains were grown separately in following four media i) LB, ii) LBT: containing 1 mg/ml of tryptophan iii) AMM, iv) AMMT: containing 1 mg/ml of tryptophan. LB and AMM were used as a control media and LBT and AMMT were used for IAA production. Eight Acinetobacter genospecies were inoculated in all 4 media and incubated at 120 rpm at 28° C. up to 48 h. The wheat seeds were surface sterilized by 2% HgCl₂ (Subba Rao, 1988) and washed with sterile distilled water for 6 to 7 time to remove HgCl₂ completely. After washing, seeds were added in above-mentioned culture and kept on shaker at 120 rpm for 2 h at 28° C. After 2 h, wheat seeds were aseptically collected and inoculated in pots containing sterile soil. Wheat seeds mixed with uninoculated media as well as with distilled water were treated as control. The pots were kept in sunlight and raised under close supervision. The growth of plants was observed everyday for 21 days. After 21 days plants were carefully uprooted and root and shoot lengths were measured. Same experiment was done simultaneously using large size pots up to 4 months for complete life cycle of wheat plant.

Statistical Analysis:

Root length (RL) and shoot length (SL) were considered as main parameters to find out effect of IAA on wheat plant. Statistical analysis was done with the help of mean, standard deviation and analysis of variance (ANOVA) (Kulkarni et al, 1999).

Plasmid Isolation:

Eight strains were checked for presence of plasmid(s). Plasmid/s were isolated by three different methods described by Kado and Lui (1981), Sambrook et al (1989) and Bimboim and Doly (1979). The presence of plasmid(s) was tested by 0.7% agarose gel electrophoresis in TAE buffer at 52 V for 6 to 8 h. Ethidium bromide stained gels were observed under gel documentation system (Alpha Imager™ 2200 Documentation and Analysis System, Alpha Innotech Corporation, California, USA) and photographed. Molecular weight was determined by comparing with 1 kb DNA ladder.

Plasmid Curing:

Plasmid curing was done for all eight strains of Acinetobacter genospecies using ethidium bromide (1024 μg/ml) and heat (52° C.) as described by Deshpande & Chopade (1994).

Transformation:

E. coli HB 101 (rif^(r)) mutant was used as a recipient for transformation. Plasmid DNA isolated from A. haemolyticus (A19) was used for transformation of DNA as this strain showed good IAA production and also exhibits other interesting characteristics such as resistance to selenium, lead and tellurium (which were used as genetic markers for plasmid transformation), chitinase production and antimicrobial activity against plant as well as human pathogenic fungi and bacteria (Huddedar & Chopade, 2000). Transformation was carried out by preparing E. coli HB101 (rif^(r)) mutant cells competent by CaCl₂ method (Sambrook et al, 1989) and competent E. coli cells were mixed with plasmid pUPI126 DNA (10 μl). The transformants were selected and checked for IAA production by Salkowaski test and parent recipient E. coli HB101 (rif^(r)) mutant was used as control.

Results:

Isolation, Identification and Confirmation of Acinetobacter Genospecies:

Colonies showing mucoid character were selected and tested for gram character and morphology, motility, presence of capsule, oxidase and catalase production. Gram negative, coccobacilli, non motile, oxidase negative, catalase positive and capsulated strains were considered as tentative Acinetobacter spp and confirmed by chromosomal DNA transformation assay. 37 Acinetobacter strains isolated from five stages of wheat plant were confirmed as genuine Acinetobacters. Acinetobacter strains were identified to species level on the basis of biochemical tests. These Acinetobacters were not biotypable by API 20NE system. Eight IAA producing strains were identified as A. baumannii (A18, A16, A13 and A30), A. haemolyticus (A19), A. junii (A6) and A. genospecies 3 (A15, A28) (Table 1).

Production of IAA by Acinetobacter Strains:

All 37 Acinetobacters were tested for IAA production by nitrocellulose paper assay. It was observed that among 37 Acinetobacter strains only 8 Acinetobacter strains showed red ring around growth of bacterial colony within 1 min on addition of Salkowaski reagent. IAA production for these eight Acinetobacter strains was also detected by Salkowaski method and development of red color indicated presence of IAA. 1 ml distilled water and 4 ml of Salkowaski reagent was taken as negative control.

Time Course of IAA Producing Acinetobacter spp:

It was found that five strains of Acinetobacter such as A. haemolyticus (A19), A. baumannii (A18, A16, A13) and A. genospecies 3 (A15), showed maximum IAA production in early stationary phase (48 h). A. junii (A6) showed maximum IAA production in log phase (24 h) and A. genospecies 3 (A28) and A. baumannii (A30) showed maximum IAA production in late stationary phase (60 and 72 h). FIGS. 1A and B, represent the growth phase with maximum IAA production by each Acinetobacter spp.

Extraction and Purification of IAA by TLC:

Extraction of IAA was done by ethyl acetate. The Salkowaski test was done at each step of the extraction showed that only the organic phase contained IAA. The dry powder obtained after evaporation of ethyl acetate (Bouchi evaporater) showed presence of IAA which was further fractionated by preparative TLC. The band pattern of purified IAA was comparable with standard IAA. (FIG. 2). It was observed that when these bands from TLC were scratched and again tested by TLC, showed a single band as that of standard IAA with R_(f) value 0.5. The amount of auxin found in culture filtrate was 4 mg/Lit.

Identification of IAA by IR Spectrum and by Melting Point:

The IR spectrum of pure IAA showed OH frequency at 3384.9 cm⁻¹ and C═O frequency at 1698.4 cm⁻¹ (FIG. 3). IR spectrum of standard IAA also showed same results. The melting point of purified IAA was found to be 168° C. which is also same as that of standard IAA.

Analysis of IAA by ¹H-NMR:

The ¹H-NMR of purified eight IAA samples was found to be same as that of ¹H-NMR of standard IAA (FIG. 4). The first peak from right is of acid, of value 9.0 δ bs (—OH), lateral peaks are of protons having values 7.8 δ d 1H (C₈H), 7.5 δ d 1H (C₅H), 7.31δ d 1H (C₂H) and 7.07 δ m 2H (C₆ & C₇H). Middle big peak is of DMSO having value of 3.04 δ S (d⁶) and moisture. The next peak is of carbon and hydrogen, having value of 2.59 δ S 2H (—CH₂—) and last peak is of internal standard, tetra-methyl-silnate (TMS) of value 0.08.

Effect of pH on IAA Production:

It was observed that at acidic pH (pH 4 & 5) Acinetobacter genospecies could not grow. The growth and IAA production was observed from pH 6 to pH 9 and found that pH 7 was optimum for IAA production by A. baumannii (A16, A18, A30), A. genospecies 3 (A15), A. haemolyticus (A19), and A. junii (A6). Two strains viz A. baumannii (A13) and A. genospecies 3 (A28) showed optimum IAA production at pH 9 (Table 2).

Effect of IAA Produced by Acinetobacter on Growth of Wheat Plant:

Statistical analysis showed significant difference in root and shoot length of test wheat plants (21 days) as compared to control plants (Table 3). Similar difference was observed when the same experiment was done using large size pots (Table 4). It was observed that there was a difference in color of leaves of control and inoculated plants. Color of leaves of plants inoculated with Acinetobacter spp. was dark green as compared to pale green of control. Interestingly width of shoots of inoculated plants was found to be almost double as compared to control. It was also observed that development of inflorescence stage (flowering stage) and fruiting stages were observed 10 days earlier in inoculated plants as that of control. (FIG. 5).

Plasmid Isolation:

All eight Acinetobacter strains contained one plasmid of the molecular weight 40 kb. All three methods of plasmid DNA showed the presence of only one plasmid in all eight strains. The plasmid was designated as pUPI126.

Plasmid Curing:

Plasmid pUPI126 was not cured by ethidium bromide even at concentration of 1024 μg/ml and heat (52° C.).

Transformation of Plasmid pUPI126:

Plasmid pUPI126 showed resistance to selenium, tellurium and lead was transformed to E. coli HB101 rif^(r) mutant at the frequency of 5×10⁻⁵. Along with IAA, selenium, tellurium and lead were also co-transferred almost at the same frequency. Transformants showed IAA production as checked by Salkowaski test. The color developed was pink as compared to red, developed by original host A. haemolyticus A19 strain. The negative control, E. coli HB101(rif^(r)) mutant did not show any color formation with Salkowaski reagent. Plasmid pUPI126 was isolated from transformants and observed by 0.7% agarose gel electrophoresis.

Plasmid pUPI126 is novel. Plasmid pUPI126 encodes IAA production, along with resistance to tellurium, selenium, arsenate and kanamycin. The cultures containing plasmid pUPI126 are already submitted to the National Collection of Industrial Microorganisms (NCIM), at National Chemical Laboratory (NCL), Pune, India. These cultures are easily available for experiment. Names and NCIM numbers of these strains are as follows: Name of Bacteria NCIM Number I. A. haemolyticus A19 (pUPI126) NCIM 5155 II. A. genospecies 3 A28 (pUPI126) NCIM 5159 III. A. genospecies 3 A15 (pUPI126) NCIM 5151 IV. A. baumannii A13 (pUPI126) NCIM 5158 V. A. baumannii A16 (pUPI126) NCIM 5156 VI A. baumannii A18 (pUPI126) NCIM 5157 VII. A. baumannii A30 (pUPI126) NCIM 5154 VIII. A. junii A6 (pUPI126) NCIM 5153

We are also going to submit above cultures to MTCC, Chandigargh, an international Depository, India.

The eight strains are novel. It is important to note that Acinetobacter spp. form rhizosphere of any plant has not been isolated and studied in detailed for its characterization. For comparison, soil isolates were taken as per reference of Bouvet and Grimont(1986, 1987) and A. calcoaceticus MTCC127. Difference between results made Acinetobacter strains distinct from known Acinetobacter. The detailed characteristics of these strains is given as follows: TABLE 1 Identification of IAA producing Acinetobacter spp isolated from rhizosphere of wheat. A. baumannii A. baumannii A. baumannii A. baumannii A. baumannii Characteristics (Std)* A13 A16 A18 A30 Growth at: 44⁰ C. ++ ++ ++ ++ ++ 41⁰ C. ++ ++ ++ ++ ++ 37⁰ C. ++ ++ ++ ++ ++ 28⁰ C. ++ ++ ++ ++ ++ Pigment production ND NP Brown Yellow NF Acid from Glucose ++ ++ NP ++ NP Gelatin Hydrolysis −− −− −− −− ++ Haemolysis −− ++ −− −− ++ Citrate (Simmons) ++ ++ ++ ++ ++ Utilization of: DL - Lactate ++ ++ ++ ++ ++ L - Phenylalanine ++ ++ ++ ++ −− Phenyl acetate ++ ++ −− ++ −− Malonate ++ ++ ++ ++ ++ L - Histidine ++ ++ ++ ++ ++ D - Malate ++ ++ ++ −− ++ L - Aspartate ND ++ ++ ++ −− L - Leucine ++ ++ ++ ++ −− L - Tyrosine ++ ++ ++ ++ −− β- Alanine ++ ++ ++ ++ ++ L - Glycine ND ++ ++ ++ ++ Trans-Aconitate ++ −− ++ ++ −− D- Glucose ND ++ ++ ++ ++ L - Tryptophane −− ++ ++ ++ ++ Na - Acetate ND ++ ++ ++ ++ Ethanol ++ ++ ++ ++ ++ L - Arginine ++ ++ ++ ++ ++ L - Ornithine ++ ++ ++ −− ++ DL 4-aminobutyrate ++ ++ ++ ++ ++ Other tests: Indole ND −− −− −− −− MR ND −− −− −− ++ Triple sugar iron test ND ++ ++ ++ ++ (std)*: From Bouvet and Grimont (1986, 1987). This was done as per Bouvet and Grimont (1986 & 1987). Besides these some additional tests were done. −−: negative, ++: positive, NP: not produced, ND: Not detected. Bold indicates differential tests.

TABLE 2 A. genospecies A. genospecies A. genospecies Characteristics 3 (Std)* A15 3 A28 Growth at: 44⁰ C. −− −− −− 41⁰ C. ++ ++ ++ 37⁰ C. ++ ++ ++ 28⁰ C. ++ ++ ++ Pigment production −− Pink Pink Acid from Glucose ++ ++ ++ Gelatin Hydrolysis −− −− −− Haemolysis −− −− −− Citrate (Simmons) ++ ++ ++ Utilization of: DL - Lactate ++ ++ ++ L - Phenylalanine ++ ++ ++ Phenyl acetate ND ++ −− Malonate ++ ++ ++ L - Histidine ++ ++ ++ D - Malate ++ ++ ++ L - Aspartate ++ ++ ++ L - Leucine ND ++ ++ L - Tyrosine ++ ++ ++ β- Alanine ++ ++ ++ L - Glycine ND ++ ++ Trans-Aconitate ++ ++ ++ D- Glucose ND ++ ++ L - Tryptophane ND ++ ++ Na - Acetate ND ++ ++ Ethanol ++ ++ ++ L - Arginine ++ ++ ++ L - Ornithine ++ ++ ++ DL 4-Aminobutyrate ++ ++ ++ Other tests: MR ND ++ ++ VP ND −− −− Triple sugar ion test ND ++ ++ (std)*: From Bouvet and Grimont (1986,1987). *This was done as per Bouvet and Grimont (1986 & 1987). Besides these some additional tests were done. −−: negative, ++: positive, NP: not produced, ND: Not detected. Bold indicates Differential test.

TABLE 3 A. haemolyticus A. A. junii A. junii Characteristics (Std)* haemolytivusA19 (std)* A6 Growth at: 44⁰ C. −− ++ −− ++ 41⁰ C. ++ ++ ++ ++ 37⁰ C. ++ ++ ++ ++ 28⁰ C. ++ ++ ++ ++ Pigment −− Yellow −− −− production Acid from ++ ++ −− −− Glucose Gelatin ++ ++ −− ++ Hydrolysis Haemolysis ++ ++ −− −− Citrate ++ ++ ++ ++ (Simmons) Utilization of: DL - Lactate −− ++ ++ ++ L - −− ++ −− ++ Phenylalanine Phenyl acetate ++ ++ −− ++ Malonate −− ++ −− ++ L - Histidine ++ ++ ++ ++ D - Malate ++ ++ ++ −− L - Aspartate −− −− ++ ++ L - Leucine −− −− −− ++ L - Tyrosine −− ++ ++ ++ β- Alanine −− ++ −− ++ L - Glycine ND ++ ND ++ Trans-Aconitate −− −− −− ++ D- Glucose ND ++ ND ++ L - ND ++ ND ++ Tryptophane Na - Acetate ND ++ ND ++ Oxalate ND ++ ND −− Ethanol ++ ++ ++ ++ L - Arginine ++ ++ ++ ++ L - Ornithine ++ ++ −− −− DL 4- ++ ++ ++ ++ Aminobutyrate Tween 20 ND ++ ND ++ Tween 80 ND ++ ND ++ Other tests: Indole ND −− ND −− MR ND −− ND −− VP ND −− ND −− Triple sugar ++ ++ ++ ++ iron test (std)*: From Bouvet and Grimont (1986, 1987). *This was done as per Bouvet and Grimont (1986 & 1987). Besides these some additional tests were done. −−: negative, ++: positive, NP: not produced, ND: Not detected. Bold indicates differential test.

From table1, it was found that at least 10 tests for Acinetobacter baumannii, isolated from rhizosphere of wheat gave different results from the standard A. baumannii. Table 2, indicates that 6 tests for A. genospecies 3, isolated from rhizosphere of wheat gave different results from standard A. genospecies 3. Table 3, indicates that 6 tests from A. haemolyticus and 8 tests for A. junii, isolated from rhizosphere of wheat gave different results from standard A. haemolyticus and A. junji respectively. Thus on the basis of these new characteristics these strains are novel.

The invention of the instant Application involve an inventive step because though microbes in the rhizosphere of plants are able to produce IAA, it is not necessary that each microbe is going to produce IAA. In case of Acinetobacter, we studied the presence and role of Acinetobacter in rhizosphere of wheat and proved experimentally that Acinetobacter is significantly present in the rhizosphere of wheat. Significant presence of Acinetobacter motivated us to find the role of Acinetobacter in the rhizosphere of wheat. After lot of experimental work we proved that Acinetobacter is able to produce IAA and production of IAA is encoded by plasmid pUPI126 which is the first report on plasmid encoded IAA production in the genus Acinetobacter.

The Microbes are Used as Bioinoculant.

Following experiment was done to prove that Acinetobacter is used as bioinoculant. This is additional work which we have done for confirmation of effect of IAA produced by Acinetobacter on wheat plant.

i. Preparation of Acinetobacter Bioinoculum:

Acinetobacter bioinoculum was prepared by using lignite as a carrier. Lignite was sterilized at 121° C. for 1 h. After 1 h autoclaving lignite was kept for cooling. A. genospecies 3 A28 wild & mutant (A28.1) and A. haemolyticus A19 wild and rif^(r) mutant (A19.1) were inoculated in 500 ml of Luria broth and flasks were incubated at 120 rpm for 48 h at 30° C. After 48 h flasks were removed, sterile lignite and cultures of A. genospecies 3 A28 wild and rifampicin resistant mutant as well as A. haemolyticus A19 wild and rif^(r) mutant were mixed separately in the proportion of 500 mg lignite and 250 ml of each culture, aseptically using sterile gloves. The mixture of each bioinoculum was labeled properly and incubated at 30° C. for 24 h in sterile plastic bags in two sets. These sets were then used in field treatments of bioinoculum to wheat plants. (Subba Roq, 1988).

ii. Field trials of Acinetobacter Bioinoculum for Wheat Plant:

Field trials of novel bioinoculum of A. genospecies 3 A28 wild and rif^(r) mutant (A28.1) as well as A. haemolyticus A19 wild and rif^(r) mutant (A19.1) were taken, Acinetobacter bioinoculum was used for the field trial on the wheat plant. Variety of wheat plant was HD 2189 and bioinoculum and chemical fertilizer treatments were given in two different fields, first: experimental field of Mahatma Phule Agriculture College, Shivajinagar, Pune and second: a field of a local farmer at Hadapsar, Pune. Following types of the treatments were given to the wheat seeds with and without application of bioinoculum and by using different doses of chemical fertilizers:

-   -   a) Treatment 1: Wheat seeds were treated with only bioinoculum         of A. genospecies 3 A28 wild.     -   b) Treatment 2: Wheat seeds were treated with only bioinoculum         of A. haemolyticus A19 wild.     -   c) Treatment 3: Wheat seeds were treated with only bioinoculum         of A. genospecies 3 A28 mutant.     -   d) Treatment 4: Wheat seeds were treated with only bioinoculum         of A. haemolyticus A19 mutant.     -   e) Treatment 5: Wheat seeds were treated with bioinoculum of A.         genospecies 3 A28 wild and half dose of fertilizer as in         step (b) above.     -   f) Treatment 6: Wheat seeds were treated with bioinoculum of A.         haemolyticus A19 wild and half dose of fertilizer as in step (b)         above.     -   g) Treatment 7: Wheat seeds were treated with bioinoculum of A.         genospecies 3 A28 mutant and half dose of fertilizer as in         step (b) above.     -   h) Treatment 8: Wheat seeds were treated with bioinoculum of A.         haemolyticus A19 mutant and half dose of fertilizer as in         step(b) above.     -   i) Treatment 9: Wheat seeds treated with standard Azotobacter         bioinoculum (provided by Mahatma Phule Agriculture College,         Shivajinagar, Pune).     -   j) Treatment 10: Wheat seeds treated with standard Azotobacter         bioinoculum. (provided by Mahatma Phule Agriculture College,         Shivajinagar, Pune) with half dose of fertilizer as in step (b)         above.     -   k) Treatment 11: Wheat seeds treated with standard Azospirillum         bioinoculum. (provided by Mahatma Phule Agriculture, College,         Shivajinagar, Pune).     -   l) Treatment 12: Wheat seeds treated with standard Azospirillum         bioinoculum. (provided by Mahatma Phule Agriculture, College,         Shivajinagar, Pune) with half dose of fertilizer as in step (b)         above.     -   m) Control 1: Without bioinoculum and chemical fertilizer.     -   n) Control 2: Without bioinoculum and with half dose of chemical         fertilizer such as Urea 0.6 kg/100 m², and single super         phosphate 1.3 kg/100 m².

After these treatments wheat plants were observed for different growth parameters.

iii. Effect of Colonization of Acinetobacter to the Wheat Plants in the Field:

Colonization study is important to indicate effect of Acinetobacter bioinoculum is effectively colonize the roots of wheat plant. This confirms the plant growth promoting effect of Acinetobacter bioinoculum on wheat plant.

Rif^(r) mutants isolated from A. genospecies 3 A28.1 and A. haemolyticus A19.1 were mainly used for this purpose. Colonization by these mutants was observed at 4 major life stages of wheat plants, after: i. 30 days, ii. 45 days, iii. 60 days and iv.75 days. At all these 4 life stages of wheat plants treated with only mutants of A. genospecies 3 A28 and A. haemolyticus A19 were uprooted and the roots were washed thoroughly by sterile distilled water for 5-6 times. Roots were then cut into small pieces of 2-3 cm, weighed 1 g and kept in sterile saline (0.85%) then kept on shaker for half an hour. After that serial dilution was made in the sterile saline and dilutions were then plated out on Luria agar containing 100μg/ml of rifampicin. Plates were incubated at 28° C. for 48-60 h. Colony count was taken after 60 h. This experiment was done in duplicate. The colonization of wheat root was also carried out by root mapping method (Brown, 1962) and wheat roots were mapped on Luria agar with 100 μg/ml of rifampicin plates and incubated at 28° C. for 24-48 h and observed for colonization.

iv. Effect of Acinetobacter Bioinoculum on Wheat Plant:

Effect of Acinetobacter bioinoculum on wheat plant was observed considering following parameters.

-   -   a. Measurement of shoot length of wheat plants (total height of         plant).     -   b. Measurement of number of tillers of wheat plants &     -   c. Measurements of width of leaves of wheat plants.

Significant difference between controls and different treatments was calculated by employing statistical methods such as, Mean, Standard Deviation, ANOVA and T-test.

The inventiveness of the invention can be further substantiated by the fact that it is not true that, Pseudomonas is producing IAA that's why Acinetobacter is producing it.

In fact, it was fist time, we proved that genus Acinetobacter is also able to produced IAA and takes part in plant growth promotion. Behavior of Pseudomonas is not a motivation/clue for us, In fact, in order to understand role of Acinetobacter in plant growth promotion, we proposed a hypothesis. This hypothesis was prove experimentally by us. For this about 800 references were studied in detail. By studying biology of Acinetobacter from literature and our previous large number of publications on Acinetobacter, we formulated a hypothesis entitled “Acinetobacter spp. may be present in the rhizosphere of wheat and may have important properties. It may exhibit characteristics like antibiotic and metal resistance, antibiotic, bioemulsifier and IAA production to promote plant growth. It may carry plasmids encoding some of the special features and it may help to understand the role of Acinetobacter in rhizosphere of wheat.” Lot of creativity, hard work, thinking and experimental planning has made it possible to prove this hypothesis.

*−: Negative, +: Positive. TABLE 4 Effect of pH on IAA production by Acinetobacter genospecies. A. genospecies 3 A. baumannii A. junii A. haemolyticus pH A15 A28 A16 A18 A30 A13 A6 A19 6 0.02 0.08 0.07 0.81 1.23 0.06 1.46 1.22 7 0.13 0.14 0.32 1.10 1.90 1.45 1.43 1.95 8 0.11 0.23 0.21 0.92 1.12 1.46 1.35 1.01 9 0.10 0.35 0.29 0.58 0.88 1.52 1.28 0.55 * OD measured at 540 nm. * At pH 4 and 5 there was no growth of Acinetobacter genospecies hence there was no IAA production.

TABLE 5 Effect of IAA producing A. genospecies on root length and shoot length of 21 days wheat plant by ANOVA Test. Root Length (cm) Shoot Length (cm) Source Df SS MS F df SS MS F Medium 4 2193.7 548.4 33.2 4 3407.8 851.9 42.5 Bacteria 8 3900.4 487.5 29.5 8 1770.0 221.2 11.0 Interaction 24 3814.7 158.9 9.6 24 2669.7 111.2 5.5 Error 1069 17622.6 16.4 648 154726.4 20.0 Total 1105 27,531.4 684 df: degree of freedom, SS: sum of squares, MS: mean squares, F: F-test., Medium: AMM, LB, AMMT and LBT, Bacteria: Acinetobacter genospecies, Interaction: Interactions takes place between bacteria—bacteria & media and bacteria.

TABLE 6 Effect of IAA produced by Acinetobacter genospecies on root and shoot length of wheat plant at ripened fruiting stage (75d)^(a). Root length (cm) Shoot length (cm) Genospecies Mean SD Mean SD A. baumannii A18 36.7 5.02 40.1 0.1 A13 32.3 2.5 36.9 5.7 A30 32.4 4.8 35.1 4.8 A16 37.2 2.3 33.5 5.5 A. haemolyticus A19 35.3 3.5 41.7 2.8 A. junii A6 35.6 2.5 40.1 0.1 A. genospecies 3 37.2 4.3 40.3 0.3 A15 41.4 2.1 42.6 2.08 A28 ^(a)Values are based on three sets of experiments. SD: Standard Deviation.

Acinetobacter is commonly found in soil (Baumann,1968; Dhakephalkar & Chopade 1994^(b)).Therefore it was logical to believe that it may be present in rhizosphere. However there is no report on the presence of Acinetobacter in the rhizosphere of plants including wheat plant, which is an economically important plant. There is only a passing statement on the presence of Acinetobacter in the wheat rhizosphere (Kleeberger et al, 1983). Acinetobacter is relatively recently studied group of microorganisms. This is because previously this bacteria was known by 40 different names and hence there has been a lot of confusion about the systematics of Acinetobacter spp. (Henriksen, 1973). With the development of chromosomal DNA transformation assay by Juni (1972 & 1978), a genuine method of confirmation of Acinetobacter was developed. This assay is specific only for Acinetobacter genospecies and based on this assay the authenticity of genus Acinetobacter was established (Juni, 1972 & 1978). At present the systematics of Acinetobacter is well defined and it consists of 21 genospecies (John, et al 1994, Bouvet & Grimont 1986 & 1987). Aim of this study was to find out the effect of IAA production by Acinetobacter strains on the overall life cycle of wheat plant.

Our work in this area was done systematically because of routine use of chromosomal DNA transformation assay for confirmation of genus Acinetobacter (Juni, 1972; Deshpande & Chopade, 1994). Interestingly all 37 isolates of Acinetobacter were confirmed by this assay. It is important to note that, out of 21 genospecies, only five Acinetobacter genospecies were detected from rhizosphere of wheat. Our findings have revealed that Acinetobacter is present in the rhizosphere of wheat in significant number (Huddedar & Chopade, 2000). This finding gave us the most valuable clue that Acinetobacter may have some role in the wheat rhizosphere. It should be noted that Acinetobacter is not a plant pathogen. To find out its role in the plant growth promotion, we did screening of Acinetobacter spp for IAA production. It is important to note that until now there is no report on the production of IAA in the genus Acinetobacter from wheat rhizosphere. Determination of IAA producing capacity of microorganism is useful in its identification and provided a valuable marker when examining physiological role or ecological significance of IAA in establishment and persistence of organism in the rhizosphere (Bric et al, 1988). As compared to other IAA producing bacteria, production of IAA with respect to growth phase of Acinetobacter is similar that it produced IAA in stationary phase, but A. junii (A6) produced IAA in log phase. Interestingly the IAA production by Acinetobacter is qualitatively strong as it takes dark red color within one min. when it reacts with Salkowaski reagent on the nitrocellulose paper.

The TLC of extracts clearly showed presence of IAA in all eight Acinetobacter genospecies (FIG. 2). The ¹H-NMR, IR and melting point of extracted samples matched with the standard IAA. We found that pH also affected the IAA production and neutral pH (7) was found to be best for production of IAA for four Acinetobacter genospecies which were, A. genospecies 3, A. baumannii, A. junji and A. haemolyticus. (A15, A16, A18, A13, A6 and A19). Out of eight, in two Acinetobacter genospecies, A. baumannii A13 and A. genospecies 3 A28, IAA was produced in maximum amount at alkaline pH as compared to acidic pH. This fact has ecological significance as, pH of clay soil used for cultivation of wheat in Maharashtra, India, is alkaline (pH 8 to 10). The effect of IAA on plant root, shoot length and width, fruiting capacity and health of the plants as compared with control plant clearly indicated that IAA is produced by Acinetobacter and it is directly involved in plant growth promotion. The Acinetobacter genospecies grown in AMMT or LBT promoted maximum growth of wheat plants in pots since the growth media was supplemented with tryptophan, the precursor for IAA production. While the Acinetobacter strains grown in AMM and LB promoted less growth of plants, as there was lack of tryptophan. This observation indicates that plant growth promotion was definitely by IAA produced from Acinetobacter genospecies. Similarly the effect of an inoculation with IAA producing 3 Pseudomonas and 1 Acinetobacter on root growth resulted in increased shoot growth of maize plant has been demonstrated (Lippmann et al, 1995).

Plasmid isolation and transformation of plasmid pUPI126 to E. coli HB101 (Rif^(r)) mutant provided evidence that production of IAA and resistance to selenium, tellurium and lead genes are encoded on the plasmid pUPI126 in Acinetobacter haemolyticus A19 and E. coli HB101 transformants also produced IAA in stationary phase. Our findings are very similar to previously published results of Pseudomonas savastanoi in which IAA producing genes are also encoded on plasmid pIAA1 (Comai & Kosuge,1982; Costacurta & Vanderleyden, 1995). To the best of our knowledge this is the first report of the IAA production in the genus Acinetobacter. It is observed that plasmid pUPI126 was not cured either by ethidium bromide or heat which suggests that this plasmid is very stable in its original host Acinetobacter. Plasmid transfer and behavior is well established in Acinetobacter (Chopade et al, 1985; Deshpande & Chopade 1994; Naik et al, 1994). It would be worth to investigate the behavior of this plasmid pUPI126 in the rhizosphere microorganisms.

Besides indole acetic acid (IAA) encoded by plasmid pUPI126, other characteristics such as production of hormone/s like cytokine as it has been reported in plasmid pP4TH in Erwinia herbicola pv. Phypsophiloe (Clark, et al, 1993) are encoded by plasmid and this warrants further investigation. This work has indeed established the role of Acinetobacter in wheat rhizosphere. It is expected that this work will provide stimulus to the work on Acinetobacter plant interactions in variety of economically important plants. Present study has successfully shown the effect of IAA on the growth of wheat plant. Large size pot experiments have confirmed this observation. Besides enhancement of growth of wheat plant, the flowering (inflorescence) and fruiting stages of life cycle were reached about 10 days earlier and persisted longer than the control. Overall, health of wheat plant was very much improved as compared to control. This indicates the potential of Acinetobacter as a novel bioinoculant for wheat. Further studies on cloning of IAA genes and its regulation, pathway of IAA biosynthesis and field studies on effect of IAA produced by different Acinetobacter genospecies on growth and yield of wheat plant and development of bioinoculant are in progress.

Conclusions:

This is the first report on plasmid encoded IAA production in genus Acinetobacter. From this result the role of Acinetobacter in wheat rhizosphere becomes very clear. It proves that Acinetobacter has symbiotic interaction with wheat plant and able to stimulate plant growth. Thus this bacteria can be further used to increase in yield of wheat plant.

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1. A plasmid pUPI126 encoding indole-3 acetic acid (IAA) production.
 2. A plasmid as claimed in claim 1, wherein the plasmid is of 40 Kb.
 3. A plasmid as claimed in claim 1, wherein the plasmid encodes resistance to selenium, tellurium, and lead.
 4. Acinetobacter strains having plasmid pUPI126 encoding indole-3 acetic acid (IAA) production.
 5. The strains as claimed in claim 3, wherein the strains are selected from a group comprising Acinetobacter haemolyticus A19, Acinetobacter genospecies A28, Acinetobacter genospecies A15, Acinetobacter baumannii A13, Acinetobacter baumannii A16, Acinetobacter baumannii A18, Acinetobacter baumannii A30, and Acinetobacter junii A6.
 6. The strains as claimed in claim 3, wherein the plasmid encodes resistance to selenium, tellurium, and lead.
 7. The strains are claimed in claim 3, wherein the strain Acinetobacter haemolyticus A19 is characterized as: Characteristics A. haemolytivusA19 Growth at: 44⁰ C. ++ 41⁰ C. ++ 37⁰ C. ++ 28⁰ C. ++ Pigment production Yellow Acid from Glucose ++ Gelatin Hydrolysis ++ Haemolysis ++ Citrate (Simmons) ++ Utilization of: DL - Lactate ++ L - Phenylalanine ++ Phenyl acetate ++ Malonate ++ L - Histidine ++ D - Malate ++ L - Aspartate −− L - Leucine −− L - Tyrosine ++ β- Alanine ++ L - Glycine ++ Trans-Aconitate −− D- Glucose ++ L - Tryptophane ++ Na - Acetate ++ Oxalate ++ Ethanol ++ L - Arginine ++ L - Ornithine ++ DL 4-Aminobutyrate ++ Tween 20 ++ Tween 80 ++ Other tests: Indole −− MR −− VP −− Triple sugar iron test ++


8. The strains are claimed in claim 3, wherein the strain Acinetobacter genospecies A28 is characterized as: Characteristics A. genospecies 3 A28 Growth at: 44⁰ C. −− 41⁰ C. ++ 37⁰ C. ++ 28⁰ C. ++ Pigment production Pink Acid from Glucose ++ Gelatin Hydrolysis −− Haemolysis −− Citrate (Simmons) ++ Utilization of: DL - Lactate ++ L - Phenylalanine ++ Phenyl acetate −− Malonate ++ L - Histidine ++ D - Malate ++ L - Aspartate ++ L - Leucine ++ L - Tyrosine ++ β- Alanine ++ L - Glycine ++ Trans-Aconitate ++ D- Glucose ++ L - Tryptophane ++ Na - Acetate ++ Ethanol ++ L - Arginine ++ L - Ornithine ++ DL 4- ++ Aminobutyrate Other tests: MR ++ VP −− Triple sugar ion ++ test


9. The strains are claimed in claim 3, wherein the strain Acinetobacter genospecies a15 is characterized as: Characteristics A. genospecies A15 Growth at: 44⁰ C. −− 41⁰ C. ++ 37⁰ C. ++ 28⁰ C. ++ Pigment production Pink Acid from Glucose ++ Gelatin Hydrolysis −− Haemolysis −− Citrate (Simmons) ++ Utilization of: DL - Lactate ++ L - Phenylalanine ++ Phenyl acetate ++ Malonate ++ L - Histidine ++ D - Malate ++ L - Aspartate ++ L - Leucine ++ L - Tyrosine ++ β- Alanine ++ L - Glycine ++ Trans-Aconitate ++ D- Glucose ++ L - Tryptophane ++ Na - Acetate ++ Ethanol ++ L - Arginine ++ L - Ornithine ++ DL 4-Aminobutyrate ++ Other tests: MR ++ VP −− Triple sugar ion test ++


10. The strains are claimed in claim 3, wherein the strain Acinetobacter baumannii A13 is characterized as: Character A. baumannii A13 Growth at: 44⁰ C. ++ 41⁰ C. ++ 37⁰ C. ++ 28⁰ C. ++ Pigment production NP Acid from Glucose ++ Gelatin Hydrolysis −− Haemolysis ++ Citrate (Simmons) ++ Utilization of: DL - Lactate ++ L - Phenylalanine ++ Phenyl acetate ++ Malonate ++ L - Histidine ++ D - Malate ++ L - Aspartate ++ L - Leucine ++ L - Tyrosine ++ β- Alanine ++ L - Glycine ++ Trans-Aconitate −− D- Glucose ++ L - Tryptophane ++ Na - Acetate ++ Ethanol ++ L - Arginine ++ L - Ornithine ++ DL 4-aminobutyrate ++ Other tests: Indole −− MR −− Triple sugar iron test ++


11. The strains are claimed in claim 3, wherein the strain Acinetobacter baumannii A16 is characterized as: Character A. baumannii A16 Growth at: 44⁰ C. ++ 41⁰ C. ++ 37⁰ C. ++ 28⁰ C. ++ Pigment production Brown Acid from Glucose NP Gelatin Hydrolysis −− Haemolysis −− Citrate (Simmons) ++ Utilization of: DL - Lactate ++ L - Phenylalanine ++ Phenyl acetate −− Malonate ++ L - Histidine ++ D - Malate ++ L - Aspartate ++ L - Leucine ++ L - Tyrosine ++ β- Alanine ++ L - Glycine ++ Trans-Aconitate ++ D- Glucose ++ L - Tryptophane ++ Na - Acetate ++ Ethanol ++ L - Arginine ++ L - Ornithine ++ DL 4-aminobutyrate ++ Other tests: Indole −− MR −− Triple sugar iron test ++


12. The strains are claimed in claim 3, wherein the strain Acinetobacter baumannii A18 is characterized as: Character A. baumannii A18 Growth at: 44⁰ C. ++ 41⁰ C. ++ 37⁰ C. ++ 28⁰ C. ++ Pigment production Yellow Acid from Glucose ++ Gelatin Hydrolysis −− Haemolysis −− Citrate (Simmons) ++ Utilization of: DL - Lactate ++ L - Phenylalanine ++ Phenyl acetate ++ Malonate ++ L - Histidine ++ D - Malate −− L - Aspartate ++ L - Leucine ++ L - Tyrosine ++ β- Alanine ++ L - Glycine ++ Trans-Aconitate ++ D- Glucose ++ L - Tryptophane ++ Na - Acetate ++ Ethanol ++ L - Arginine ++ L - Ornithine −− DL 4-aminobutyrate ++ Other tests: Indole −− MR −− Triple sugar iron test ++


13. The strains are claimed in claim 3, wherein the strain Acinetobacter baumannii A30 is characterized as: Character A. baumannii A30 Growth at: 44⁰ C. ++ 41⁰ C. ++ 37⁰ C. ++ 28⁰ C. ++ Pigment production NF Acid from Glucose NP Gelatin Hydrolysis ++ Haemolysis ++ Citrate (Simmons) ++ Utilization of: DL - Lactate ++ L - Phenylalanine −− Phenyl acetate −− Malonate ++ L - Histidine ++ D - Malate ++ L - Aspartate −− L - Leucine −− L - Tyrosine −− β- Alanine ++ L - Glycine ++ Trans-Aconitate −− D- Glucose ++ L - Tryptophane ++ Na - Acetate ++ Ethanol ++ L - Arginine ++ L - Ornithine ++ DL 4-aminobutyrate ++ Other tests: Indole −− MR ++ Triple sugar iron test ++


14. The strains are claimed in claim 3, wherein the strain Acinetobacter junii A6 is characterized as: Characteristics A. junii A6 Growth at: 44⁰ C. ++ 41⁰ C. ++ 37⁰ C. ++ 28⁰ C. ++ Pigment production −− Acid from Glucose −− Gelatin Hydrolysis ++ Haemolysis −− Citrate (Simmons) ++ Utilization of: DL - Lactate ++ L - Phenylalanine ++ Phenyl acetate ++ Malonate ++ L - Histidine ++ D - Malate −− L - Aspartate ++ L - Leucine ++ L - Tyrosine ++ β- Alanine ++ L - Glycine ++ Trans-Aconitate ++ D- Glucose ++ L - Tryptophane ++ Na - Acetate ++ Oxalate −− Ethanol ++ L - Arginine ++ L - Ornithine −− DL 4-Aminobutyrate ++ Tween 20 ++ Tween 80 ++ Other tests: Indole −− MR −− VP −− Triple sugar iron test ++


15. A bioinoculum for wheat plant, said bioinoculum comprising one or more strain selected from a group consisting of Acinetobacter haemolyticus A19, Acinetobacter genospecies A28, Acinetobacter genospecies A15, Acinetobacter baumannii A13, Acinetobacter baumannii A16, Acinetobacter baumannii A18, Acinetobacter baumannii A30, and Acinetobacter junii A6; and a carrier.
 16. A bioinoculum as claimed in claim 15 wherein the carrier is lignite.
 17. A method of promoting wheat plant growth, said method comprising treating wheat seeds with the bioinoculum.
 18. A method as claimed in claim 17, wherein the bioinoculum comprising one or more strain selected from a group consisting of Acinetobacter haemolyticus A19, Acinetobacter genospecies A28, Acinetobacter genospecies A15, Acinetobacter baumannii A13, Acinetobacter baumannii A16, Acinetobacter baumannii A18, Acinetobacter baumannii A30, and Acinetobacter junii A6; and a carrier.
 19. A method as claimed in claim 15 wherein the carrier is lignite.
 20. A method as claimed in claim 15 wherein the Acinetobacter colonizes during the treatment of the seed.
 21. A method as claimed in claim 15 wherein the color of the leaf becomes darker green.
 22. A method as claimed in claim 15 wherein the inflorescence and fruiting stages of the plant is reached in time duration lessened by 10 days. 